In the present studies, we have investigated the modulation of atrial natriuretic factor (ANF) receptor of R2 subtype (ANF-R2) coupled to adenylyl cyclase/cAMP signal transduction system by angiotensin II (AII). C-ANF4-23 [C-ANF4-23, [des(Gln18, Ser19, Gln20, Leu21, Gly22)ANF4-23-NH2] and AII inhibited adenylyl cyclase activity in control vascular smooth muscle cells (VSMC A-10) by about 40% and 30% respectively. Pretreatment of the cells with AII resulted in the attenuation of both C-ANF4-23- and AII-mediated inhibition of adenylyl cyclase. Losartan treatment of the cells was able to partially block (approximately 50%) the AII- as well as C-ANF4-23-mediated inhibitions of adenylyl cyclase that are completely lost by AII pretreatment. The pretreatment of the cells with AII alone or with losartan did not affect the [125I]-ANF binding to ANF receptors. However, AII treatment resulted in the augmentation of the levels of Gi alpha 2 and Gi alpha 3. On the other hand, staurosporine (a protein kinase C [PKC] inhibitor) treatment of cells before AII treatment was able to prevent the attenuation of both C-ANF4-23 as well as AII-mediated inhibition of adenylyl cyclase elicited by AII. These results indicate that the AII modulation of ANF-R2 receptor-mediated inhibition of adenylyl cyclase is independent of ANF-R2 receptor density or the levels of Gi regulatory protein and may be due to the uncoupling of the ANF-R2 receptor from the Gi protein. This uncoupling may be associated with the phosphorylation of the Gi protein by PKC activated by AII.